Effect of heme binding efficiency with recombinant protein on activity of RhDyPB

Abstract:

Background Dye-decolorizing peroxidases (DyPs) are members of the heme peroxidase family, and their catalytic activities depend on a non-covalently bound heme cofactor. DyPs are able to degrade a variety of toxic and harmful dyes, serving as promising biocatalysts. However, the low efficiency of heme binding to the apo-enzyme in the production of recombinant DyPs limits their catalytic activities.Objective To enhance the binding efficiency of heme with the apo-enzyme, we selected mutation sites based on structural information analysis and molecular docking to improve the enzyme activity.Methods Mutagenesis was performed on RhDyPB derived from Rhodococcus jostii RHA1 to obtain the high-activity mutant E156K, which bound more heme cofactors. The enzymatic properties and kinetic parameters of E156K were analyzed, and the binding capacity of RhDyPB and E156K to heme was evaluated.Results The heme binding capacity and specific activity of E156K were 14.14 nmol/mg and 19.5 U/mg, respectively, which were 57.5% and 56.0% higher than those of RhDyPB. This indicated higher heme binding capacity had a positive impact on the catalytic activity of this enzyme, while the optimal pH and temperature had no obvious difference between E156K and RhDyPB. Finally, the catalytic activity of E156K was further enhanced through fermentation condition optimization, 50.3% higher than that without optimization.Conclusion This study enhances the catalytic efficiency of RhDyPB by rational design based on the heme binding capacity, providing a basis for the molecular modification and industrial application of DyPs.